Small biopsy fixatives and their applications

Fixatives used in histopathology for small biopsies

1. SMALL BIOPSY FIXATIVES AND THEIR APPLICATIONS Presenter : Dr. Revathi Krishnamurthy Moderator : Dr. Seema Bijjaragi

2. FIXATION It is a process by which the constituents of cells are fixed in a physical and partly chemical state so that They will withstand subsequent treatment with various reagents Minimal distortion of tissue organisation or decomposition Tissue is preserved in as life-like manner as possible

3. PURPOSE OF FIXATION To prevent bacterial degeneration, autolysis and putrefaction To maintain as close a resemblance as possible to the natural structure of tissue components To withstand chemicals used at various stages of processing Clear staining of sections Permit the cutting of thin slices of tissue Increase optical differentiation of cellular structures

4. IDEAL FIXATIVE Good tissue penetration Stabilises the tissue, preserving its character and cellular distribution Prevents fixation artifacts Prevents structure deformation Safe to handle – nontoxic and nonallergenic

5. FIXATIVES Simple fixatives Compound fixatives 10% Formalin Glutaraldehye Ethyl alcohol Formalin based 10% neutral buffered formalin 10% neutral buffered formol saline Formol calcium Dichromate fixatives Regaud’s solution Moller’s solution Orth’s solution Mercurial fixatives Zenker’s solution Helly’s solution B5 fixative reagent Picric acid fixatives Bouin’s solution Gendre’s solution Alcohol containing Carnoy’s fluid Acetic alcohol formalin

6. Based on their mode of action Physical methods : Heating, microwaving, freeze drying Chemical methods : Aldehydes (cross-linking): Formaldehyde, Glutaraldehyde , Mercurial fixatives Oxidising agents : Osmium tetroxide, chromate containing fixatives Protein denaturation (coagulants/ dehydrants ) : Acetic acid, Methyl alcohol, Ethyl alcohol

7. ALDEHYDES

8. FORMALDEHYDE 10% formalin Formalin - 10 ml Water - 90 ml Neutral buffered formalin Formalin - 100ml Water - 900ml Sodium phosphate monobasic monohydrate – 4gm Sodium phosphate dibasic anhydrous – 6.5gm

9. PRINCIPLE Cross-linking of protein molecules by aldehyde group Methylene bridges Reactive hydroxymethyl side groups Reversible on washing formalin

10. 10 – 20 times its volume 24 hours at room temperature Small : 8 – 12 hours How much ?

11. Advantages Easily available and cheap Relatively stable (especially if buffered) Application of most staining techniques without special preliminary procedures Does not cause excessive tissue hardening Preservation of fats, myelin, nerve fibres, amyloid Best fixative for nervous system !!

12. Disadvantages Formalin pigment Becomes cloudy on storage Not suitable for electron microscopy due to its denaturing effect on proteins Irritation to the eyes and causes dermatitis Formalin pigment associated with RBCs in a section of kidney

13. GLUTARALDEHYDE Available as 25% or 50% stock solution pH : 3 – 5 Temperature : 0 - 4º C Amber coloured : - Acrolein - Glutaric acid - Ethanol Fixation : - 2.5% - 4% Small tissue fragments Needle biopsies Fixation time : 2-4 hours At room temperature

14. PRINCIPLE Dialdehyde Stabilises protein structure by cross-linking Irreversible

15. Advantages Electron microscopy : Fixes tissues rapidly and stabilises proteins Fixes small tissue fragments and needle biopsies in 2-4 hours at room temperature Better preservation of cellular and plasma proteins than formaldehyde Better ultrastructural preservative than formaldehyde Fixation remains potent for 3 months at 0-4º C Less shrinkage of tissues as compared to formalin Less irritating to handle

16. Disadvantages More expensive Slow penetration – hence tissues must be small Not suitable for carbohydrates, lipids and immunohistochemical methods Not recommended as a fixative when PAS is likely to be required

17. MERCURIALS

18. ZENKER’S FLUID Distilled water : 100 ml Mercuric chloride : 5 g Potassium dichromate : 2.5 g Sodium sulphate : 1 g 5 ml glacial acetic acid to be added just before use Biopsies of : Lymph node Spleen Bone marrow Congested tissues Thymus Fixation time: Average-sized blocks : 12 hours Small biopsies : 30 – 60 min

19. HELLY’S FLUID Same composition as that of Zenker’s fluid 5 ml of formalin is added immediately before use Biopsies of: Bone marrow Spleen Extramedullary hematopoiesis Fixation time : 8 – 24 hours

20. B5 FIXATIVE REAGENT Stock reagent A Mercuric chloride - 12g Sodium acetate - 2.5g Distilled water - 200ml Stock reagent B 10% neutral buffered formalin 90 ml 1 0 ml

21. Biopsies of : Bone marrow Lymph node Fixation time 8 – 12 hours

22. HEIDENHAIN’S “ SUSA ” FIXATIVE Mercuric chloride : 45g Sodium chloride : 5g Formalin : 200ml Distilled water : 800ml Glacial acetic acid : 40ml Trichloroacetic acid : 20g Many laboratories in Europe prefer this fixative for small biopsies Minimal shrinkage Rapid fixation Excellent staining

23. Capable of decalcifying very small fragments of bone Fixation time: Small biopsies – 2-3 hours Very small fragments – 1 hour

24. Note! The tissue must be washed overnight to remove excess dichromate Mercuric chloride pigment must be removed : Iodine solution : 5 – 10min Sodium thiosulfate

25. Advantages Best results with metachromatic stains Routine fixatives of choice for preservation of detail for photography !! Zenker’s fluid Helly’s fluid Better staining of nuclei and connective tissues Preserves cytoplasmic granules Trichrome stains Giemsa / Leishman stains

26. Disadvantages Solutions of mercuric chloride corrode metals Mercuric chloride causes marked shrinkage The complete solution deteriorates rapidly Zenker’s solution causes lysis of red blood cells and removes much iron from hemosiderin Tissues become unduly hard and brittle when left in these fixatives for more than 1-2 days

27. Disadvantages: Formation of diffuse black granules – Mercury pigment A section of kidney that shows randomly distributed black deposits

28. DICHROMATE FIXATIVES

29. Principle Oxidation of proteins Reduced chromate ions cause cross-linking of proteins Fixatives containing chromate at a pH of 3.5 – 5 are good fixatives, which make proteins insoluble even without coagulation

30. REGAUD’S FLUID Potassium dichromate - 3g Distilled water - 80ml Add 20ml of 10% formalin at the time of use ORTH’S FLUID Potassium dichromate - 2.5g Sodium sulphate - 1g Distilled water - 90ml Add 10ml formaldehyde at the time of use

31. To demonstrate: C hromaffin tissue Mitochondria Golgi apparatus Mitotic figures Colloid-containing tissues Red blood cells

32. Disadvantages Darkens on standing due to acidity Bleaching of tissue pigments on prolonged fixation Glycogen fixation is poor

33. POSTCHROMATION When chromation (oxidation) is desired in tissues that have already been fixed in other fixatives Placed in 2.5% potassium dichromate Before processing After processing 6-8 days 12-24 hours

34. Renders phospholipids more resistant to extraction by dehydrating and clearing agents Employed to mordant tissues for staining, particularly mitochondria, thus helping in improved staining and preservation

35. PICRIC ACID FIXATIVES

36. BOUIN’S FLUID Picric acid saturated aqueous solution - 75ml Formalin - 25ml Glacial acetic acid - 5ml Fixation time 1 to 12 hours

37. Advantages Penetrates rapidly and evenly Little shrinkage Brilliant staining with : - Masson’s Trichrome - Giemsa - Mallory stains Good fixative for glycogen as well as connective tissue

38. Biopsies of : Liver Muscle Testis Gastrointestinal tract Endocrine tissue

39. Disadvantages Lyses RBCs Reduces the amount of demonstrable iron Tissues become hard and brittle on prolonged fixation Lipids are altered

40. Note ! They precipitate proteins and form protein picrates , which are yellow in colour C an be removed through subsequent changes in 50-70% alcohol Hence, tissues should not be placed in water directly after fixation To remove yellow colour on sections: Saturated solution of lithium carbonate in 70% ethyl alcohol Ethyl alcohol followed by 5% sodium thiosulfate, wash in running water

41. GENDRE’S FLUID Picric acid saturated solution in 95% alcohol - 80ml Formalin - 15ml Glacial acetic acid - 5ml Better than Bouin’s fluid for glycogen and connective tissue!

42. ALCOHOL CONTAINING FIXATIVES

43. Alcohol causes denaturation of proteins and changes their physical properties, making them insoluble CARNOY’S FIXATIVE Absolute alcohol - 60ml Chloroform - 30ml Glacial acetic acid - 10ml

44. Advantages Penetrates rapidly Very suitable for small tissue fragments like curettings - fixation in ½ to 2 hours Initiates dehydration Good preservative for glycogen Good Nuclear staining Hemorrhagic samples

45. Disadvantages Shrinkage of cells Loss of chromatin material D issolves lipids and myelin Note ! Must be prepared fresh when needed and discarded after each use

46. FIXATION OF INDIVIDUAL TISSUES

47. TYPES OF BIOPSY Closed indirect biopsy Core needle biopsy Punch biopsy Loop biopsy Endoscopic biopsy FNAB Open direct biopsy Incisional biopsy Excisional biopsy Closed image guided biopsy Stereotactic biopsy USG guided CT guided MRI guided

48. Renal biopsy 2 cores are obtained whenever possible Light microscopy  First core Immunofluorescence Electron microscopy Light microscopy Neutral buffered formalin Electron microscopy 3% glutaraldehyde Immunofluorescence Transported to laboratory either: Fresh (saline soaked gauze) Zeus or Michael medium Second core

49. Muscle biopsy Biopsies of muscle are transported to the laboratory in a saline-moistened gauze Light microscopy Neutral buffered formalin Histochemistry Deep frozen Electron microscopy 3% glutaraldehyde

50. Liver biopsy Routine 10% Neutral buffered formalin Suspected glycogen storage disorder Carnoy’s fixative

51. Lymph node biopsy: Neutral buffered formalin B5 fixative Testicular biopsy Neutral buffered formalin Bouin’s fluid Helly’s fluid

52. Nerve biopsy: Specimen is transported to the laboratory in a damp gauze Light microscopy 10% Neutral buffered formalin Electron microscopy 4% glutaraldehyde Biochemical studies Snap frozen Immunofluorescence studies

53. Brain biopsy: May be divided or assorted for different procedures depending upon the amount of tissue sent and urgency of the report required Light microscopy  Formalin fixation (1-2 hours) Cryostat sections  Freezing in liquid nitrogen - Flash frozen in isopentane cooled by liquid nitrogen at -160ºC - Rapid diagnosis - Biochemical investigations

54. Electron microscopy  2.5% glutaraldehyde (4 hours) Squash preparation  Acetic alcohol

55. Bone marrow biopsy Fixatives Decalcifying agent Embedding Neutral buffered formalin 18 – 24h EDTA Paraffin Bouin’s solution <24 h EDTA B5 <24 h EDTA Schaffer’s solution 4 - 16 h Formic acid Nitric acid 12 – 24h 6 – 18h Plastic Zinc formalin

56. Routine 10% Neutral buffered formalin Immunofluorescence Zeus or Michael’s medium Skin biopsy

57. Bladder/ureter biopsy Bouin’s fluid Formalin GI biopsy Bouin’s fluid Formalin Breast biopsy Formalin Amyloidosis Formalin Alcohol Gouty tophus Alcohol

58. CONCLUSION Proper tissue fixation is essential to ensure the highest level of specimen evaluation Once a tissue has been fixed inadequately or inappropriately, remedial changes may no longer be possible Most often formalin is an adequate choice, if not the optimal one. However, in certain situations placing the tissue in formalin may limit the ability to reach a definitive diagnosis It is imperative for the pathologists to know and communicate which fixative is optimal

59. REFERENCES Grizzle WE, Fredenburgh JL, Meyers RB. Fixation of Tissues. In: Suvarna SK, Layton C, Bancroft JD, editor. Theory and Practice of Histological Techniques, 7 th ed. China: Elsevier; 2013. p. 69-94. Culling CFA. Fixation of Tissues. In: Lynch MJ, editor. Medical Laboratory Technology and Clinical Pathology, 4 th ed. Philadelphia: W.B.Saunders Co, 1983. p. 876-89 Shariff S, Kaler AK. Fixation. In: Shariff S, editor. Principles & Interpretation of Laboratory Practices in Surgical Pathology, 1 st ed. New Delhi: Jaypee Brothers Medcial Publishers. p. 1-17 Rai R, Bhardwaj A, Verma S. Tissue Fixatives: A review. Int J. Pharm. Drug. Anal, 2016;4:183-7 Qidwai K, Afkhami M, Day CE. The Pathologist’s guide to fixatives. In: Day CE, editor. Histopathology methods and Protocols, 1 st ed. New York: Springer,2014. p. 21-30